Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca+2 mobilizing 2nd messenger. This application proposes a collaborative effort to synthesize novel and informative NAADP analogs and to apply these compounds to solve important problems in the field-including the unambiguous identification of the NAADP binding domain, a through elucidation of the structure-activity relation for NAADP recognition, an appreciation for the variation in the SAR between species and between tissues, and the production of potent, pharmacologically well characterized and membrane permanent agonists and antagonists. Evidence strongly suggests that NAADP regulates the two-pore channel (TPC) located on a lysosomal-like acidic organelle, and that NAADP mediated Ca+2 release may serve as a trigger to initiate the release of Ca+2 ion, which is then amplified through other Ca+2 release mechanisms. A variety of tissues and cell types have been shown to respond to NAADP, arguing for the importance and generality of this signaling mechanism. NAADP derivatives will be synthesized using chemo-enzymatic methods as well as through total synthesis. First, we propose to apply a photoaffinity label developed in the PI's laboratory to label NAADP binding proteins in mammalian cell extracts and in sea urchin homogenates, and to determine if the NAADP binding domain is a domain of the TPC, the ryanodine receptor, or if it is a novel protein. Purification and isolation of the photoderivatized binding protein will be facilitated by our synthesis of potent bifunctional photoprobes containing an aromatic azide for photocrosslinking to the receptor as well as an acetylenic substituent that will be used after photocrosslinkage to attach an affinity tag using click chemistry. This procedure will enable us to purify the small quantities of photoderivatized NAADP binding protein and subsequently to identify the binding protein by obtaining partial sequences using mass spectrometry. NAADP analogs will be characterized using sea urchin egg homogenates and in mammalian cells using patch clamp and single cell fluorescent measurements, enabling us to characterize the behavior of our compounds both in the important invertebrate system and in mammalian cells. Last, we will synthesize and test metabolically stable NAADP derivatives. Compounds demonstrated to be high potency agonists and antagonists will be converted into acytoxymethyl esters and developed as membrane permanent agonists and antagonists. PUBLIC HEALTH RELEVANCE: Changes in of intracellular calcium-ion concentration are a fundamental cell regulatory mechanism. Nicotinic acid adenine dinucleotide 2'-phosphate (NAADP) is one of several interacting intracellular 2nd messengers that mobilize calcium-ion to control biological response. Results of this program will enhance our understanding of NAADP signaling mechanisms, identify new therapeutic targets, and uncover novel drug strategies. It will contribute to understanding fertilization, insulin secretion, smooth muscle contraction, and other important processes.